To reduce high attrition rates and accelerate discovery and development timelines, the pharmaceutical industry seeks in vitro alternatives to interrogate drug candidates prior to animal, humanized animal and human studies. Enormous cost, ethical concerns and increased pressure from regulatory agencies to replace, reduce and refine animal use in drug discovery and toxicity testing all drive demand for cell-based models that can be used for in vitro screening to triage toxic and ineffective leads earlier, prior to in vivo studies. However, it has been difficult finding a cell-based model that not only mimics the function of living tissues but that can also be appropriately analyzed in the drug screening context.
Cost-efficient interrogation of drug candidates often requires high throughput methodology. Cell monolayers, or a layer of cells growing, for example, in a petri dish, were commonly used in cell-based assays and were optimal for high throughput screening processes. However, it was soon found that cell monolayers had a very low success rate in predicting in vivo therapeutic outcomes of drug candidates.
The poor results obtained with cell monolayers have been attributed to the vast differences in the monolayer cell environment and the in vivo cell environment. Cell morphology, extracellular matrix interactions, three-dimensional organization, oxygen tension and access to both the therapeutics being tested and other extracellular factors all differ greatly between cells found in a monolayer and cells found in vivo.
Recently, three-dimensional (3D) cell cultures have emerged as an alternative to a flat layer of cells as a means to model tissues with improved physiological relevance for biomedical research and in vitro drug testing of all stages. 3D cell cultures are cellular networks in which cells are round and organized in three dimensions, an environment and cell morphology that are more similar to that found in vivo. Examples include three-dimensional cell aggregates such as tumor spheroids, embryoid bodies and hanging drop cell cultures; cultures grown in three-dimensional scaffolds; and the cultures grown in extracellular matrix gels or gels mimicking the extracellular matrix, among others. According to Comley (Comley, D. J., 2010. Drug Discovery World. 11(3): 25-41), the formats that showed the most promise in 3D cell culturing were gel/hydrogel, followed by ECM (extracellular matrix) sheet, aggregates/spheroids, and then collagen tissue constructs. This study was based on industry-wide global market survey results from 78 university/research institute/not-for-profit facilities, 28 biotech companies, 11 others, nine hospitals/clinics, seven pharmaceutical companies, four government/military/defense facilities, two fee-for-service providers, one biomanufacturing/bioprocessing lab, one diagnostics company, and one agrochemical company.
The Comley article describes that most of the prior art advances in 3D cell culture revolved around the use of a hydrogel or collagen scaffold (termed a biomimetic scaffold) (Comley, J., Drug Discovery World, Summer, pp. 25-41, 2010.) Gel/hydrogel based formats have become favored by the industry because they are better able to mimic the cellular environment found in tissues in vivo. When grown in extracellular matrices or gels functionalized to mimic extracellular matrix, the cells are round and interact with the other cells and the matrix in a manner that is closer to the in vivo situation. Oxygen tension, extracellular availability of soluble factors, cell adhesion molecules and the stiffness of the extracellular environment can be adjusted by adjusting the gel composition to reflect those in native tissues. Two other platforms for 3D cell culture being pursued by prior art researchers were structural scaffolds and microfluidic devices, structural scaffolds being defined as materials made from the same material as 2D plate surfaces such as polystyrene and having some 3D microstructure.
Despite advances made by many prior art researchers, the Comley article describes that most prior art 3D cell culture applications were riddled with problems and failed to meet the needs of their consumers. The article specifically notes that these prior art compositions and methods provided a “[v]irtual lack of proven automated solutions,” and that “all methods need[ed] higher throughput.” The study went on to note that there was “poor reproducibility between batches of biometric scaffolds,” there was a “limited ability to scale up or down a single 3D format,” and that “better visualization [and] wider applicability to HCS [(high content screening)]” was needed.
Low production control, low throughput, difficult handling, and incompatibility with high-throughput screening and high-content screening readouts remain key limitations for widespread adoption of gel-based 3D cell culture models as a mainstream approach in routine screening workflows. The four most significant problems with using gel-based cultures for drug screening are described below.
First, sol-state gels comprising cells are difficult to plate so that characteristic culture dimensions are consistent and reproducible and it is difficult to do so in sufficient throughput needed in pharmaceutical testing. For those gels having a gelling mechanism that depends on temperature, gelling may start during culture dispensing at plating. This produces culture-to-culture variations in culture shape and height for the same volume of dispensed sol-state extracellular matrix (ECM) gel plus cells due to temperature variations during both dispensing and gelling. An example of such gel is BD™ MATRIGEL™.
Second, cellular distribution within the gel is non-uniform in three dimensions and/or difficult to control. This is true for various types of gels and gelling mechanisms including but not limited to thermo-reversible gels (i.e., the BD™ MATRIGEL™), gels requiring physiological temperature to initiate or accelerate polymerization (i.e., Collagen type I), gel precursors which gel by addition of cross-linking agents (i.e., Glycosan HYSTEM™) and those which self-assemble (i.e., BD™ PURAMATRIX™), among others. Regardless of the gelling method, whether mediated by chemical or physical cross-linking, cells settle if gelling is slow. Typical gelling time scale is at the order of tens of minutes, during which time cells may be settling. This leads to non-uniform cell distribution within gel 3D cultures, which is further exacerbated by variations in environmental parameters causing additional culture-to-culture inconsistencies.
Inconsistent culture dimensions and cell density across these dimensions yield culture-to-culture inconsistent supply of nutrients, removal of catabolic waste products and intra-culture concentrations of trophic factors, autocrine and paracrine signaling molecules cells secrete to regulate their environment, growth and many other functions. Some signaling molecules degrade quickly, limiting the scope of their effectiveness to the immediate cell surroundings. Others affect only nearby cells because they are taken up quickly, or because their transport is hindered by the extracellular matrix. For these reasons, inconsistent cultures at plating are more likely to yield inconsistent tissue analogs for drug testing. Variations in cell function and secretion of drug metabolizing enzymes influence pharmacokinetic and pharmacodynamic studies, resulting in potentially less conclusive cellular outcomes and problems with interpretation of results.
Third, gels are flexible and not suitable for vigorous experimentation and routine handling. Even at low throughput levels, the prior art tissue reconstructions are too delicate for routine handling, assaying and screening, especially after days in culture, a period which is required for 3D cell cultures to mature into functional tissue reconstructions for drug testing in the same. During this period in culture, gels may deteriorate before cells secrete and reconstitute their own endogenous matrix support, or gels may become too frail so that a culture disintegrates on routine pipetting. While gels degrade by a variety of mechanisms depending on their chemistry, presence of cells and environment, they typically break down (lose mass or dissolve) by mechanisms that reverse gelling, such as enzymatic or hydrolytic degradation or cell-mediated matrix remodeling and digestion, among other factors.
Fourth, temporal variations in gel composition and its structural integrity contribute to variability in three-dimensional gel-based cell culturing. This variability is present batch-to-batch, and even culture-to-culture, in the same batch owing to susceptibilities to environmental factors that govern gelling and due to matrix remodeling by cells, which is different culture-to-culture due to inadequate control of cell distribution at plating.
Each of these issues likely contributes to the findings of the Comley article relating to the lack of appropriate three-dimensional cell cultures for high-throughput screening and high-content screening applications. Accordingly, what are needed in the art are 3D cell culture compositions and methods of using said compositions to transform inconsistent, low experimental count 3D cultures into consistent and reliable high-throughput screening and high-content screening diagnostic tools. More specifically, what are needed are imaging-accessible 3D cell culture compositions and methods of using the same to reproducibly plate consistent cultures in high-throughput; routinely culture, handle, apply test agents and assay said cultures in high-throughput; and routinely screen in said cultures, days and weeks after culture plating, using standard high-throughput screening and high-content screening tools.